1. Field of the Invention
This invention relates to DNA regions and their combinations which are particularly useful for inclusion in recombinant DNA vectors for the expression of inserted genes, especially genes encoding the light (L) and heavy (H) chains of an antibody molecule.
The invention further relates to chimeric antibodies with human tumor cell specificity and their derivatives, nucleotide and protein sequences coding therefor, as well as methods of obtaining and manipulating such sequences.
2. Background
The expression of genetically engineered proteins from mammalian cells provides materials useful for the diagnosis and treatment of human and veterinary diseases and disorders. Examples of such proteins include tissue plasminogen activator, erythropoietin, hepatitis B surface antigen, and genetically engineered antibodies. Mammalian cells, such as chinese hamster ovary or hybridoma cells, provide convenient hosts for the production of many such proteins because of their ability to properly glycosylate, assemble, fold, and secrete the engineered protein. These qualities make mammalian cells particularly useful for the production of antibody molecules, which are glycosylated multimeric proteins consisting of two identical H chains combined with two identical L chains in a specific three-dimensional molecular arrangement.
Several gene expression systems for the production of genetically engineered proteins from mammalian cells have been developed. These systems include vectors designed for either the transient or permanent expression of the desired gene when introduced into the host cell. Many of these vehicles include DNA regions or elements which provide various gene expression functions, such as promotion of transcription initiation, transcription promoter enhancement, mRNA splicing, mRNA polyadenylation, and transcription termination. This invention describes specific gene expression elements and recombinant DNA expression vectors that are particularly useful for the production of genetically engineered antibodies from mammalian cells.
The majority of reported applications of genetically engineered antibodies have utilized gene expression elements which accompany the immunoglobulin coding regions upon recombinant DNA molecular cloning (reviewed by Oi, V. T., and Morrison, S. L., Biotechniques 4:214 (1986)). A chimeric mouse-human antibody will typically be synthe-sized from genes driven by the chromosomal gene promoters native to the mouse H and L chain variable (V) regions used in the constructs; splicing usually occurs between the splice donor site in the mouse J region and the splice acceptor site preceding the human constant (C) region and also at the splice regions that occur within the human H chain C region; polyadenylation and transcription termination occur at native chromosomal sites downstream of the human coding regions. Some of these gene expression elements, particularly the transcription promoters, are unpredictable because of their differing origins from one antibody V region gene sequence to the next. This unpredictability may be an impediment to the efficient expression of a chosen recombinant immunoglobulin gene, as noted for some chimeric L chains by Morrison, S. et al., Proc. Natl. Acad. Sci., USA 81:6851 (1984) (p.6854). A convenient alternative to the use of chromosomal gene fragments is the use of cDNA for the construction of chimeric immunoglobulin genes, as reported by Liu et al. (Proc. Natl. Acad. Sci., USA 84:3439 (1987) and J. Immunology 139:3521 (1987)). The use of cDNA requires that gene expression elements appropriate for the host cell be combined with the gene in order to achieve synthesis of the desired protein. This property could help overcome the unpredictability of recombinant antibody synthesis through the use of specific gene expression elements, such as viral transcriptional promoter sequences, to uniformly achieve efficient antibody synthesis. Although many gene expression elements have been tested in various systems, there are few studies on gene expression elements for recombinant immunoglobulin cDNA genes. There is therefore a substantial need for identification of improved gene expression elements and their combinations which are particularly suited for the efficient synthesis of genetically engineered antibody proteins by desired host cells. Gene expression elements that have been used for the expression of cDNA genes include:
(i) Viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama, H. and Berg, P., Mol. Cell. Biol. 3:280 (1983)), Rous sarcoma virus LTR (Gorman, C. et al., Proc. Natl. Acad. Sci., USA 79:6777 (1982)), and Moloney murine leukemia virus LTR (Grosschedl, R., and Baltimore, D., Cell 41:885 (1985))
(ii) Splice regions and polyadenylation sites such as those derived from the SV40 late region (Okayama and Berg, supra), and
(iii) Polyadenylation sites such as in SV40 (Okayama and Berg, supra).
Immunoglobulin cDNA genes have been expressed as described by Liu et al., supra, and Weidle et al., Gene 51:21 (1987). The expression elements used for immunoglobulin cDNA gene expression were the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancers, SV40 late region mRNA splicing, rabbit xcex2-globin intervening sequence, immunoglobulin and rabbit xcex2-globin polyadenylation sites, SV40 polyadenylation elements. For immunoglobulin genes comprised of part cDNA, part chromosomal gene (Whittle et al., Protein Engineering 1:499 (1987)), the transcriptional promoter is human cytomegalovirus, the promoter enhancers are cytomegalovirus and mouse/human immunoglobulin, and mRNA splicing and polyadenylation regions are from the native chromosomal immunoglobulin sequences. Host cells used for immunoglobulin cDNA expression include mouse hybridoma (Sp2/0), monkey COS cells, and Chinese Hamster Ovary (CHO) cells. Although immunoglobulins have been successfully synthesized using these various gene expression elements and host cells, there is substantial need for improvement in the efficiency of immunoglobulin cDNA expression.
Monoclonal antibody (mAb) technology has greatly impacted current thinking about cancer therapy and diagnosis. The elegant application of cell to cell fusion for the production of mAbs by Kohler and Milstein (Nature (London) 256:495 (1975)) spawned a revolution in biology equal in impact to that of recombinant DNA cloning. MAbs produced from hybridomas are already widely used in clinical studies and basic research, testing their efficacy in the treatment of human diseases including cancer, viral and microbial infections, and other diseases and disorders of the immune system.
Although they display exquisite specificity and can influence the progression of human disease, mouse mAbs, by their very nature, have limitations in their applicability to human medicine. Most obviously, since they are derived from mouse cells, they are recognized as foreign protein when introduced into humans and elicit immune responses. Similarly, since they are distinguished from human proteins, they are cleared rapidly from circulation.
Technology to develop mAbs that could circumvent these particular problems has met with a number of obstacles. This is especially true for mAbs directed to human tumor antigens, developed for the diagnosis and treatment of cancer. Since many tumor antigens are not recognized as foreign by the human immune system, they probably lack immunogenicity in man. In contrast, those human tumor antigens that are immunogenic in mice can be used to induce mouse mAbs which, in addition to specificity, may also have therapeutic utility in humans. In addition, most human mAbs obtained in vitro are of the IgM class or isotype. To obtain human mAbs of the IgG isotype, it has been necessary to use complex techniques (e.g. cell sorting) to first identify and isolate those few cells producing IgG antibodies. A need therefore exists for an efficient way to switch antibody classes at will for any given antibody of a predetermined or desired antigenic specificity.
Chimeric antibody technology, such as that used for the antibodies described in this invention, bridges both the hybridoma and genetic engineering technologies to provide reagents, as well as products derived therefrom, for the treatment and diagnosis of human cancer.
The chimeric antibodies of the present invention embody a combination of the advantageous characteristics of mAbs. Like mouse mAbs, they can recognize and bind to a tumor antigen present in cancer tissue; however, unlike mouse mAbs, the xe2x80x9chuman-specificxe2x80x9d properties of the chimeric antibodies lower the likelihood of an immune response to the antibodies, and result in prolonged survival in the circulation through reduced clearance. Moreover, using the methods disclosed in the present invention, any desired antibody isotype can be combined with any particular antigen combining site.
The following mAbs were used to produce the chimeric antibody embodiments of this invention:
(a) the B38.1 mouse mAb (described in U.S. Pat. No. 4,612,282) was obtained from a mouse which had been immunized with cells from a human breast carcinoma, after which spleen cells were hybridized with NS-1 mouse myeloma cells. The antibody binds to an antigen which is expressed on the surface of cells from many human carcinomas, including lung carcinomas (adeno, squamous), breast carcinomas, colon carcinomas and ovarian carcinomas, but is not detectable in the majority of normal adult tissues tested. B38.1 is of the IgG1 isotype and does not mediate detectable antibody-dependent cellular cytotoxicity (ADCC) of antigen-positive tumor cells by human peripheral blood leukocyte effector cells.
(b) the Br-3 mouse mAb (Liao, S. K., et al., Proc. Am. Assoc. Cancer Res. 28:362 (1987) (where it was designated as BTMA8); Cancer Immunol.Immunother. 28:77-86 (1989)) was obtained from mice which had been immunized with cells from a human breast carcinoma, after which spleen cells were hybridized with NS-1 mouse myeloma cells. The antibody binds to an antigen which is expressed on the surface of cells from many human carcinomas, including lung carcinomas (adeno, squamous), breast carcinomas, colon carcinomas and ovarian carcinomas, but is not detectable in the majority of normal adult tissues tested. Br-3 is of the IgG1 isotype and mediates low level ADCC of antigen-positive tumor cells.
(c) the Co-1 mouse mAb (Oldham et al., Mol. Biother. 1:103-113 (1988); Avner et al., J. Biol. Resp. Modif. 8:25-36 (1989); Liao et al. (Cancer Immunol. Immunother. 28:77-86 (1989)) was obtained from a mouse which had been immunized with cells from a human colon carcinoma, after which spleen cells were hybridized with NS-1 mouse myeloma cells. The antibody binds to an antigen which is expressed on the surface of cells from many human carcinomas, including lung carcinomas (adeno, squamous), breast carcinomas, colon carcinomas and ovarian carcinomas, but is not detectable in the majority of normal adult tissues tested. Co-1 is of the IgG3 isotype and mediates ADCC of antigen-positive tumor cells.
(d) the ME4 mouse mAb (Liao, S. K., et al., J. Natl. Cancer Inst. 74:1047-1058 (1985)) was obtained from a mouse which had been immunized with cells from a human melanoma. The antibody binds to an antigen which is expressed at the surface of cells from many human melanomas and carcinomas (including lung carcinomas breast carcinomas, colon carcinomas, and ovarian carcinomas), but is not detectable in the majority of normal adult tissues tested. ME4 is of the IgG1 isotype and does not mediate ADCC of antigen-positive tumor cells.
(e) the KM10 mouse mAb (Japanese first patent publication No. 61-167699; Japanese Patent application No. 60-8129)) was obtained from a mouse immunized with an immunogen prepared from a human gastric adenoma-derived cell line, MKN-45, after which spleen cells were hybridized with P3U1 mouse myeloma cells. KM10 is of-the IgG1 isotype and binds to an antigen which is expressed on the surface of cells from many human carcinomas, including colon, stomach, pancreas and esophagus, but is not detectable in the majority of normal adult tissues tested. The hybridoma producing mAb KM10 was deposited at the Institute for Fermentation, Osaka (IFO) in Osaka, Japan on Mar. 24, 1989 under accession number IFO 50187.
The invention is directed to a combination of gene expression elements, and recombinant DNA vectors containing these elements, useful for the expression of immunoglobulin light chain and heavy chain cDNA genes in a desired host mammalian cell.
In one embodiment, for expression of cDNA genes in rodent cells, the transcriptional promoter is a viral LTR sequence, the transcriptional promoter enhancers are either or both the mouse immunoglobulin heavy chain enhancer and the viral LTR enhancer, the splice region contains an intron of greater than 31 bp, and the polyadenylation and transcription termination regions are derived from the native chromosomal sequence corresponding to the immunoglobulin chain being synthesized.
In other embodiments, cDNA sequences encoding other proteins are combined with the above-recited expression elements to achieve expression of the proteins mammalian cells.
The invention can be used to construct recombinant DNA expression vehicles to achieve efficient synthesis of antibodies in transfected host cells. Preferably, such a vehicle is constructed by the ligation of a gene expression module, containing the elements recited above, to antibody coding cDNA sequences to form a recombinant DNA molecule. Hosts, such as Sp2/0 hybridoma or Chinese Hamster Ovary cells, are then transfected with this recombinant DNA.
The invention provides engineered chimeric antibodies of desired V region specificity and C region properties, produced after gene cloning and expression of L and H chains. The chimeric antibody and its derivatives have applicability in the treatment and diagnosis of human cancer. The cloned immunoglobulin gene products and their derivatives can be produced in mammalian or microbial cells.
The invention provides cDNA sequences coding for immunoglobulin chains comprising a human C region and a non-human, V region. The immunoglobulin chains are both H and L.
The invention provides sequences as above, present in recombinant DNA molecules in vehicles such as plasmid vectors, capable of expression in desired prokaryotic or eukaryotic hosts.
The invention provides host cells capable of producing the chimeric antibodies in culture and methods of using these host cells.
The invention also provides individual chimeric immunoglobulin chains, as well as complete assembled molecules having human C regions and mouse V regions with specificity for human tumor cell antigens, wherein both V regions have the same binding specificity.
Among other immunoglobulin chains and/or molecules provided by the invention are:
1. An antibody with monovalent specificity for a tumor cell antigen, i.e., a complete, functional immunoglobulin molecule comprising:
(a) two different chimeric H chains, one of which comprises a V region with anti-tumor cell specificity, and
(b) two different L chains, with the corresponding specificities as the V regions of the H chains. The resulting hetero-bifunctional antibody would exhibit monovalent binding specificity toward human tumor cells.
2. Antibody fragments such as Fab, Fabxe2x80x2, and F(abxe2x80x2)2.
Genetic sequences, especially cDNA sequences, coding for the 3aforementioned combinations of chimeric immunoglobulin chains are also provided herein.
The invention also provides for a genetic sequence, especially a cDNA-sequence, coding for the V region of desired specificity of an antibody molecule H and/or L chain, linked to a sequence coding for a polypeptide different than an immunoglobulin chain (e.g., an enzyme). These sequences can be assembled by the methods of the invention, and expressed to yield mixed-function molecules.
The use of cDNA sequences is particularly advantageous over genomic sequences (which contain introns), in that cDNA sequences can be expressed in bacteria or other hosts which lack appropriate RNA splicing systems.